ABSTRACT

A precise balance between proliferation and differentiation must be maintained during neural development to obtain the correct proportion of differentiated cell types in the adult nervous system. The basic helix-loop-helix (bHLH) transcription factors known as E proteins and their natural inhibitors, the Id proteins, control the timing of differentiation and terminal exit from the cell cycle. Here we show that progression into S phase of human neuroblastoma cells is prevented by E proteins and promoted by Id2. Cyclin-dependent kinase inhibitors (CKI) have been identified as key effectors of cell cycle arrest in differentiating cells. However, p57Kip2 is the only CKI that is absolutely required for normal development. Through the use of global gene expression analysis in neuroblastoma cells engineered to acutely express the E protein E47 and Id2, we find that p57Kip2 is a target of E47. Consistent with the role of Id proteins, Id2 prevents activation of p57Kip2 expression, and the retinoblastoma tumor suppressor protein, a known Id2 inhibitor, counters this activity. The strong E47-mediated inhibition of entry into S phase is entirely reversed in cells in which expression of p57Kip2 is silenced by RNA interference. During brain development, expression of p57Kip2 is opposite that of Id2. Our findings identify p57Kip2 as a functionally relevant target recruited by bHLH transcription factors to induce cell cycle arrest in developing neuroblasts and suggest that deregulated expression of Id proteins may be an epigenetic mechanism to silence expression of this CKI in neural tumors.

Proper development of an organism is the result of an integrated network of differentiation programs and signaling pathways that control cell cycle exit. The timely ordered expression of tissue-specific genes is executed by transcription factors of the basic helix-loop-helix (bHLH) family (30). Class I bHLH are also known as E proteins and include E12 and E47 (two splice variants of the E2A gene), HEB, and E2-2 and are widely expressed in most mammalian tissues. They are obligate partners of class II, tissue-specific bHLH transcription factors. Heterodimerization of class I and class II bHLH requires the HLH domain, whereas DNA binding is mediated by a stretch of conserved basic amino acid residues adjacent to the HLH motif (30). The basic region associates with a hexanucleotide “E box” sequence on the DNA of target genes (CANNTG) (32, 33). Dimerization of E2A proteins with tissue-specific bHLH transcription factors activates expression of tissue-specific genes and leads to differentiation of several cell types, including muscle, neuronal, and pancreatic cells (26, 35, 55). The structurally related Id proteins (for “inhibitor of differentiation and/or DNA binding”) (3), which include Id1 to Id4, lack the basic region. Following binding to Id proteins, bHLH cannot contact DNA, and the result is loss of transcriptional activity and inhibition of differentiation. Thus, Id proteins are natural inhibitors of bHLH-mediated transcription.

So far, E proteins have been mostly studied in hematopoietic cells, where they frequently bind DNA as homodimers and exert essential functions for commitment of cells of the B and T lineages. Several direct-target genes of E proteins have been identified in these cell types (2, 11, 19, 20, 31, 42, 46). However, much less is known about the function of E proteins in other tissues. For example, although E proteins are viewed as obligate partners of neural-specific bHLH transcription factors (such as Neuro D, neurogenin, Mash 1, etc.), very few targets of E proteins have been proposed in the nervous system (43). Besides their widely accepted activity as regulators of tissue specific gene expression, a role of E proteins as cell cycle effectors has been proposed in several reports. Similar to myogenic bHLH proteins, E2A proteins decrease the efficiency of colony formation in NIH 3T3 fibroblasts, prevent serum-stimulated progression of the cell cycle (38), and inhibit entry into S phase in mesenchimal and hematopoietic cells (10, 13). In contrast with these findings, other authors reported stimulatory effects of E proteins on cell cycle progression (49, 61). In other studies, ectopic expression of E2A appeared to induce programmed cell death (19, 36). The controversial functional consequences of E proteins on the cell cycle parallel the divergent nature of cell cycle-specific target genes of E2A identified in different studies. Candidate targets to inhibit G1 progression include the cyclin-dependent kinase inhibitors (CKIs) p21Cip1, p16INK4A, and p15INK4B (13, 36, 39), whereas induction of cyclins (D3, D2, and A) has been proposed to mediate the stimulatory effect of E2A on G1-S progression (49, 61). In this scenario, it is arbitrary to predict the biological consequences and the molecular targets of E protein-dependent transcription in the nervous system.

In neural cells, differentiation is associated with permanent exit from the cell cycle, and E proteins, which are widely expressed, form heterodimers with neurogenic bHLH to activate programs of differentiation. Using gene expression profiling, we have identified the CKI p57Kip2 as a functional target of E47 in human neuroblastoma cells. We provide evidence that p57Kip2 is the primary effector of cell cycle block by E47 in tumor cells from the nervous system and establish a functional link with Id2 and its negative regulator, the retinoblastoma (Rb) tumor suppressor (15, 24). We finally show that the E47-Id2 pathway acts during development of the mouse brain to implement a proliferation checkpoint through expression of p57Kip2.

MATERIALS AND METHODS

Plasmid and cloning.The E47-ER construct was cloned into the pBabePuro vector backbone. Briefly, the full-length E47 moiety and the estrogen receptor moiety both were amplified by PCR using the following primers: E47 sense, 5′-CGCGGATCCATGAACCAGCCGCAGAGGATGGCG; E47 antisense, 5′-TCGTGAATTCATGTGCCCGGCGGGGTTGTG; ER sense, 5′-GTCGTCGACGAATTCACGAAATGAAATGGGTGC; ER antisense, 5′-ACGCGCGACTCAGATCGTGTTGGGGAAGC. The E47 moiety was digested with BamHI and EcoRI, the ER moiety was digested with EcoRI and SalI, and the pBabePuro vector backbone was digested with BamHI and SalI. The three pieces were then ligated together with the Rapid DNA Ligation kit (Roche). The bHLH-ER construct was cloned into the pBabePuro vector as described previously (46). As with full-length E47-ER, restriction endonucleases BamHI and SalI were used to clone the bHLH-ER construct into the pBabePuro vector.

Cell culture and transfection.Cell lines used in the study include neuroblastoma cell lines SK-N-SH, IMR-32, and LAN-1; osteosarcoma cell lines SAOS-2 and U2OS; lymphoma cell lines K562 and Raji; astrocytoma cell lines SNB19 and SF210; and telomerase (TERT)-immortalized human astrocytes. All cell lines were maintained in 10% fetal bovine serum albumin (Sigma) in Dulbecco's modified Eagle medium (Cambrex). Cells were transfected using Lipofectamine 2000 according to the manufacturer's instructions. The SK-N-SH and SF210 cell lines were stably transfected with the pBabePuro-E47-ER construct. After selection in 1.7 μg/ml puromycin (Sigma), individual colonies were expanded and assayed by Western blotting for expression levels of p57Kip2 after treatment with 4-hydroxytamoxifen (4-OHT) for 24 h.

Ad infection and microarray analysis.SK-N-SH neuroblastoma cells were plated 1 day prior to adenoviral (Ad) infection. Cells were infected with an adenovirus expressing E47 (Ad-E47) or with the parental virus adenovirus-cytomegalovirus 5-internal ribosomal entry site-green fluorescent protein (Ad-CMV5-IRES-GFP) (Ad-vect; Q-Biogene) at a multiplicity of infection of 100. The infection was allowed to proceed for 2 h prior to termination by the addition of excess medium to the dish. For microarray analysis, cells were harvested at the indicated times and RNA was extracted by the Trizol (Invitrogen) method. The gene expression analysis was performed with the HG-U133A Genechip (Affymetrix). Gene expression was quantified with Gene Spring 7 software and included the restriction filters indicated below.

siRNA-mediated knockdown of gene expression and BrdU incorporation studies.Small interfering RNA (siRNA) was purchased from Dharmacon and consisted of human CDKN1C for p57Kip2 (siGenome Smartpool reagent #M-003244-03) and nontargeting duplexes (siGenome Smartpool reagent #D-001206-13) as negative control. SK-N-SH and SF210 cells stably expressing pBabePuro E47-ER were transfected three times with 60 nM siRNA using Lipofectamine 2000 (Invitrogen). Twenty-four hours after the end of the third round of transfection, 1 μM 4-OHT was added for 24 or 48 h. Bromodeoxyuridine (BrdU) at a final concentration of 10 μM was added 2 h prior to cell fixation and immunostaining. Cells were stained with anti-BrdU antibody (mouse immunoglogulin G1 [IgG1] monoclonal; Roche) for 1 h at room temperature. Secondary antibody was donkey anti-mouse, Cy3 conjugated (Upstate Biotechnologies). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Cells were photographed and counted on an Olympus IX 70 inverted fluorescence microscope. Parallel cultures were analyzed by Western blotting.

Northern blotting for p57Kip2.RNA was isolated by the Trizol (Invitrogen) method. Twenty micrograms of total RNA was prepared and electrophoresed on an agarose-formaldehyde gel and transferred to a nylon membrane (Nytran SPC; Schleicher & Schuell). The membrane was prehybridized at 68°C in prehybridization solution (200 mM NaPO4, pH 7.0, 1 mM EDTA, pH 8.0, 25% formamide, 7% sodium dodecyl sulfate [SDS], 5× Denhardt's solution, 0.5 mg/ml tRNA). A 400-bp PvuII fragment of human p57Kip2 was used as a probe. Hybridization was carried out for 18 h at 68°C. Washes were carried out at 68°C in 0.1% SDS-0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) prior to autoradiography. For cycloheximide (CHX) experiments, SK-N-SH cells stably expressing pBabePuro-E47-ER were pretreated with either 20 μM CHX or vehicle for 1 h. After pretreatment, either 1 μM 4-OHT or vehicle was added for 2, 4, or 8 h. Cells were harvested and analyzed by Northern and Western blotting.

Luciferase assay.The 5× E-box-luciferase construct and either pBabePuro vector or pBabePuro E47-ER was transfected into SK-N-SH cells using Lipofectamine 2000 (Invitrogen). The cytomegalovirus early promoter-driven beta-galactosidase gene, pCMV-β-gal, was cotransfected for normalization. Four hours after transfection, 250 nM 4-hydroxytamoxifen (4-OHT; Sigma) or vehicle was added to the medium, and 24 h later cells were lysed in 1× cell culture lysis reagent (Promega) and examined for luciferase and beta-galactosidase.

Immunohistochemistry and double immunofluorescence.Sections from wild-type embryonic day 15.5 (E15.5) mouse brains were deparaffinized in xylene and rehydrated in a graded series of ethyl alcohol. Antigen retrieval was performed for 5 min in a decloaking chamber (Biocare Medical) in 10 mM Tris, pH 10 (for E47), or 1× Antigen Retrieval Citra Solution, pH 6.0 (BioGenex) (for p57Kip2 and Id2). After peroxidase block in 3% H2O2, slides were blocked for 30 min in 10% serum in PBS with 0.1% Triton X-100. Primary antibodies and dilutions were E47, 1:100 (Santa Cruz Biotechnologies N649); p57Kip2 Ab6, 1:67 (Neomarkers); and Id2, 1:200 (Zymed). Biotinylated secondary antibody (Vector Laboratories) was applied for 1 h at room temperature prior to washes. The avidin biotin peroxidase complex technique was used for primary antibody detection (Vectastain kit; Vector Laboratories). Staining was developed using diaminobenzidine (brown precipitate). Sections were counterstained with hematoxylin. Rabbit or mouse IgG (Vector Laboratories) and tissue from Id2−/− mice were used as controls for specificity of the staining. Double immunofluorescence of E47 and p57Kip2 and Id2 and p57Kip2 was performed using a tyramide signal amplification (TSA) system (Perkin Elmer Life Sciences, Inc.) according to the manufacturer's instructions. The final detection involved the use of TSA-fluorescein for p57Kip2 and TSA-Cy3 for E47 and Id2. Slides were mounted in Vectashield and analyzed with a Zeiss LSM510 confocal microscope using 20× and 40× objectives.

RESULTS

E47 induces growth arrest in neuroblastoma cells.The controversial reports of both positive and negative effects of E proteins on cell growth prompted us to determine the role of ubiquitously expressed bHLH in cells of neuroectodermal origin. We transfected SK-N-SH, IMR-32, and LAN-1 human neuroblastoma cells with plasmids encoding E47, E12, and E2-2 or the empty vector and scored the number of colonies after selection in G418. In all cell lines, E proteins decreased colony-forming efficiency with a more profound effect when E47 and E12 were used (Fig. 1A). The natural inhibitors of E proteins are Id proteins. Given the prominent role of Id2 in the biology of cells of neuroectodermal origin (16), we asked whether Id2 was sufficient to drive cell cycle progression in cells arrested in G0-G1 by serum deprivation. Ectopic expression of Id2 in SK-N-SH cells led to efficient entry into S phase, and this effect required the HLH region through which Id proteins form dimers with E proteins and abrogate their DNA binding ability (Fig. 1B).

Effect of E proteins and Id2 on neuroblastoma cell proliferation. (A) Triplicate plates of SK-N-SH, IMR-32, and LAN-1 were transfected with expression vectors encoding E12, E47, E2-2, or the empty vector. Colonies were scored after 18 days of selection in G418. (B) SK-N-SH cells were transfected with an expression vector encoding Id2, a mutated Id2 lacking the HLH domain (Id2ΔHLH), or the empty vector, and the percentage of cells entering S phase was measured by incorporation of BrdU.

To ask whether transcriptional activity by E47 is required for the effect on cell growth and to establish whether E47 acts by inhibiting cell cycle progression and/or inducing apoptosis, we designed an inducible form of E47 comprising human E47 fused at its carboxy terminus to the ligand-binding domain of the murine estrogen receptor (ER), E47-ER. The estrogen receptor is unable to bind estrogen yet retains normal affinity for the synthetic ligand 4-hydroxytamoxifen (4-OHT) (28). As a control, we used an inducible truncated form of E47 containing the bHLH domain of human E47 fused to ER. The bHLH-ER hybrid protein lacks the amino-terminal transactivation domains of E47 but can dimerize and bind to E-box sites. Recent work has shown that activation of E47-ER by 4-OHT leads to induction of B- and T-lymphocyte-specific target genes (46). After introduction in SK-N-SH cells, treatment with 4-OHT markedly induced transcription of a multimerized E-box construct that drives expression of luciferase (5× E-box-luciferase). Addition of 4-OHT to SK-N-SH cells transfected with the empty vector control or bHLH-ER did not activate transcription, indicating that E-box-driven transcriptional induction was mediated by activation of transcriptionally competent E47-ER by 4-OHT (Fig. 2A). Accordingly, chromatin immunoprecipitation (ChIP) from SK-N-SH-E47-ER using E47 antibody showed that E47 specifically bound to the multimerized E box in the presence but not in the absence of 4-OHT (Fig. 2B). Having established the functional competence of E47-ER in SK-N-SH cells, we determined its effect on cell growth, cell cycle progression, and apoptosis. First, we established that, similar to the expression of E47, 4-OHT-mediated activation of E47-ER inhibited colony formation (Fig. 2C). Next, we activated E47-ER with 4-OHT and evaluated the ratio of cells that underwent DNA synthesis. We found that upon treatment of SK-N-SH-E47-ER cells with 4-OHT, BrdU incorporation decreased by 50% and 90% after 8 and 36 h, respectively (Fig. 2D to E). Conversely, neither staining with DAPI nor fluorescence-activated cell sorting analysis of cells treated with 4-OHT showed any sign of apoptosis (nuclear fragmentation or sub-G1 peak, respectively) (Fig. 2D and data not shown). Taken together, these results indicate that (i) activation of transcription is required for the effect of E47 on cell proliferation, and (ii) E47 inhibits growth by preventing entry into S phase without a discernible effect on cell survival.

Activation of E47-ER in neuroblastoma cells induces E-box-mediated transcription and cell cycle arrest. (A) SK-N-SH cells were transfected with an E-box-luciferase construct and pBabe-E47-ER, pBabe-bHLH-ER, or pBabe vector. Luciferase activity was determined 36 h after transfection and addition of 4-OHT. Results are expressed as means ± standard deviations of triplicate assays normalized for transfection efficiency using β-galactosidase. (B) SK-N-SH cells stably expressing the fusion protein E47-ER or the empty vector were transfected with a plasmid encoding the E box of the immunoglobulin enhancer and treated with 4-OHT or vehicle control for 24 h. ChIP was performed using E47 antibody or normal rabbit IgG (NRIg), and precipitated DNA was amplified using primers specific for the E-box sequence or GAPDH. (C) SK-N-SH cells stably expressing either pBabe-E47-ER or pBabe vector alone were plated in triplicate and administered vehicle or 4-OHT for 18 days. The number of colonies is reported as the means ± standard deviations of the triplicate plating. (D) Activation of E47-ER induces inhibition of entry into S phase without apoptosis. SK-N-SH cells expressing either pBabe-E47-ER or pBabe vector were plated and treated with 4-OHT or vehicle for the indicated times. BrdU was added 90 min prior to the end of treatment with 4-OHT. Cells were processed for BrdU immunofluorescence (red), and nuclei were stained with DAPI (blue). (E) Quantitation of the experiment shown in panel D. The number of cycling cells is reported as the means ± standard deviations of three separate experiments. IP, immunoprecipitation.

The E47-Id2 transcriptome in neuroblastoma cells.To identify the key downstream target genes upon which E47 and Id2 converge to affect the cell cycle, we introduced E47 or Id2 in neuroblastoma cells and conducted a genome-wide analysis using high-density DNA oligonucleotide microarrays. To achieve acute expression of E47 and Id2 at comparable levels in SK-N-SH cells, we used recombinant adenoviruses encoding E47 or Id2 together with an IRES-green fluorescent protein (GFP). With a multiplicity of infection of 100, 100% of SK-N-SH cells expressed GFP 12 h after the infection. To identify genes that would account for cell cycle arrest caused by E47 and proliferation response implemented by Id2, we selected two independent protocols for the Ad-E47 and Ad-Id2 infections. Infection with Ad-E47 was performed in exponentially growing SK-N-SH cultured in 10% serum, whereas infection with Id2 was carried out in serum-starved quiescent SK-N-SH. Since expression of exogenous E47 and Id2 was present already 8 h after the infection (Fig. 3A), we selected 8 h and 20 h as optimal early and late time points for microarray expression profiles. Compared with cells infected with Ad vector in 10% serum, Ad-E47 infected cells arrested in the G1 phase of the cell cycle and displayed a neuronal differentiation response with elevation of the neuronal marker MAP-2 (Fig. 3B and C and data not shown for the cell cycle effects). Conversely, compared with Ad vector-infected cells cultured in 0.5% serum, infection with Ad-Id2 led to efficient entry into S phase within 20 h (Fig. 3D). E47 and Id2 target genes were identified using the Affymetrix U133A array. Absolute analysis of each chip was carried out using the default settings of the Affymetrix Microarray Suite 5.0 software to generate raw expression data. To produce a high-stringency list of candidate E47 and Id2 target genes, we carried out the following filtering and statistical analysis constraints using the GeneSpring 7.0 software (Silicon Genetics). (i) a direct E47 target should show at least a twofold change at 8 h and at least a threefold change at 20 h, whereas an “Id2 target” should show at least a threefold change at 20 h compared with the respective controls. We chose a more delayed selection criteria for Id2, because Id proteins do not bind DNA directly but can only affect gene expression through the inhibition of DNA binding of already bound target transcription factors. (ii) Data quality flag restriction was included such that the increased genes were retained only if the flag value was present (P) in all the E47 or Id2 experimental samples, and the decreased genes were retained only if the flag value was present (P) in the respective control samples. (iii) Expression value restriction was included such that the increased genes were retained only if the minimum raw expression value was ≥150 in all the E47 and Id2 experimental samples, and the decreased genes were retained only if the minimum raw expression value was ≥150 in the respective control samples. Using these restrictions, 48 probe sets corresponding to 40 individual genes were changed by E47 (33 genes up-regulated, 7 down-regulated; Fig. 4A), and 39 probe sets corresponding to 34 individual genes were changed by Id2 (18 up-regulated, 16 down-regulated; Fig. 4B). Next, using twofold restriction criteria, we asked whether any of the 40 E47 targets was reciprocally changed by Id2 (E47 up-regulated and Id2 down-regulated or E47 down-regulated and Id2 up-regulated) and whether any of the 34 Id2 targets was reciprocally changed by E47 (Id2 up-regulated and E47 down-regulated or Id2 down-regulated and E47 up-regulated). Eleven of the 40 E47 targets (28%) and 12 of the 34 Id2 targets (35%) satisfied these criteria. Among the E47-Id2 target genes, there was a known cell cycle regulator, p57Kip2, which is one of the three members of the Cip/Kip family of CKI, which also includes p21Cip1 and p27Kip1 (47). Interestingly, p57Kip2 is the only Cip/Kip inhibitor that is essential for mouse development, and several studies suggested that it integrates cell cycle and differentiation signals during development (4, 27, 56, 58-60). Indeed, the p57Kip2 signal was induced >10-fold by E47 and was one of the most highly E47-induced genes in our microarray analysis. Interestingly, in addition to p57Kip2, the small group of E47-Id2 targets included two other genes, IGF2 and IPL, that, like p57Kip2, belong to the human chromosome 11p15.5-imprinted cluster (Fig. 4 A and B) (5, 37). The 11p15.5-imprinted genes have crucial functions in differentiation, the cell cycle, and oncogenesis (14, 41, 50, 52). Expression of these genes was strongly induced by E47 and inhibited by Id2, and real-time quantitative RT-PCR on independent samples confirmed the microarray data (Fig. 5 and data not shown for IPL). Another gene in the 11p15.5 cluster, H19, was not present on the array. Real-time quantitative RT-PCR showed that Id2 and E47 changed its expression in a reciprocal manner (Fig. 5). These data suggest that E47 may act through a common enhancer to stimulate expression of the genes located in the 11p15.5-imprinted cluster and propose p57Kip2 as a mediator of the effects of E47 and Id2 on cell cycle progression.

Adenoviral expression of E47 and Id2 in human neuroblastoma cells. (A) Western blotting of E47 and Id2 in SK-N-SH cells harvested 8 h after infection with adeno-E47 (Ad-E47), adeno-Id2 (Ad-Id2), or adenovirus vector (Ad-vec). (B) Phase-contrast morphology of SK-N-SH cells infected with adeno-E47 or adenovirus vector demonstrates that E47 induces dendritic differentiation. (C) E47 induces expression of the neuronal somatodendritic differentiation marker MAP-2. SK-N-SH cells infected with adeno-E47 or adenovirus vector were harvested at the indicated times and assayed by Western blotting for MAP-2. (D) SK-N-SH cells were rendered quiescent by serum starvation, infected with adeno-Id2 or adenovirus vector, and labeled with BrdU. The percentage of BrdU-positive cells was determined at the indicated times after the infection.

Microarray analysis of the neuroblastoma cell line SK-N-SH after infection with adeno-E47 (Ad-E47) and adeno-Id2 (Ad-Id2). (A) Genes changed by adeno-E47 after 8 and 20 h. Genes located at the imprinted locus 11p15.5 are highlighted in boldface. (B) Genes changed by adeno-Id2 after 8 and 20 h. Genes reciprocally regulated by E47 and Id2 are indicated. Adenovirus vector, Ad-Vec.

Expression of chromosome 11p15.5 genes is reciprocally induced by E47 and repressed by Id2. Real-time PCR values of indicated transcript abundance in SK-N-SH cells infected with adeno-E47 (Ad-E47) or adenovirus vector (top panels) and adeno-Id2 (Ad-Id2) or adenovirus vector (bottom panels) for the indicated times. HPRT was used as an internal control.

p57Kip2 is a target of bHLH transcription factors and Id2.To validate further the changes of p57Kip2 expression by E47 and Id2, we performed a Northern blot analysis of SK-N-SH harvested at different times after infection with either E47 or Id2 adenovirus. These experiments showed marked and progressive elevation of p57Kip2 mRNA in E47-expressing cells that was already visible within 2 h (Fig. 6A, top panel). Conversely, p57Kip2 mRNA was down-regulated within 8 h of Ad-Id2 infection (Fig. 6A, bottom panel). Western blot analysis showed that p57Kip2 protein was increased by E47 with a similar time course and matched the expression of E47 (Fig. 6B). The expression of the other members of the Cip/Kip family, p21Cip1 and p27Kip1, was unaffected by E47. We confirmed that up-regulation of p57Kip2 by E47 was not a cell lineage-specific change or an artifact caused by the adenoviral system. Plasmid-mediated expression of E47 in LAN-1 neuroblastoma cells and TERT-immortalized human astrocytes demonstrated that p57Kip2 was strongly induced by E47, whereas expression of p21Cip1 and p27Kip1 was unchanged (Fig. 6C). Similarly, expression of E47 resulted in elevation of p57Kip2 in the glioma cell lines SNB19 and SF210 and osteosarcoma cell line U2OS (Fig. 6D). To ask whether p57Kip2 methylation, a mechanism involved in the establishment of imprinting on the chromosome 11p15.5 gene cluster, is affected by E47 expression, we introduced E47 in the lymphoid cells K562 and Raji. K562 carries a demethylated p57Kip2 gene, whereas p57Kip2 is aberrantly methylated in Raji cells (21). E47 induced p57kip2 in K562 but not Raji (Fig. 6D). These results indicate that E47 acts as an enhancer for the expression of p57Kip2 in cells of different tissue origins, and the effect is abolished by hypermethylation of the p57Kip2 gene.

Rapid induction of p57Kip2 by E47 in multiple cell lines. (A) Northern blot analysis of p57Kip2 following infection with adeno-E47 (Ad-E47) or adenovirus vector (Ad-vec) (upper panel) and adeno-Id2 (Ad-Id2) or adenovirus vector (lower panel). 28S rRNA is shown as a loading control. (B) Western blot analysis of E47, p57Kip2, p27Kip1, p21Cip1, and α-tubulin from SK-N-SH cells infected with adenovirus vector and adeno-E47 for the indicated times. (C) Western blot from LAN-1 human neuroblastoma cells and TERT-immortalized human astrocytes transfected with an expression plasmid encoding E47 or the empty vector demonstrates specific induction of p57Kip2. (D) SNB19, SF210, and U20S cells were transfected as described for panel C, whereas K562 and Raji cells were infected with adeno-E47 or adenovirus vector for 24 h before being analyzed by Western blotting.

The rapid induction of p57Kip2 by E47 suggested strongly that p57Kip2 elevation is the result of direct control of p57Kip2 gene expression by E47. We used the E47-ER fusion construct to determine whether the p57kip2 gene is a direct target of E47-mediated transcription. Addition of 4-OHT to SK-N-SH transfected with control vector or bHLH-ER did not modify the abundance of p57Kip2, whereas treatment with 4-OHT of cells transfected with E47-ER strongly activated p57Kip2 expression in the absence of any change of other cell cycle regulators (Fig. 7A). Furthermore, de novo protein synthesis was not required for E47-ER to induce transcription of p57Kip2 mRNA, because treatment of SK-N-SH-E47-ER cells with cycloheximide (CHX) did not prevent the robust induction of p57Kip2 mRNA by 4-OHT (Fig. 7B, CHX). Activation of E47-ER by 4-OHT induced expression of p57Kip2 independently of new protein synthesis in the glioma cell line SF210 as well (Fig. 7C). Finally, we analyzed responsiveness of the p57Kip2 promoter to E47 using a series of fragments of the promoter inserted into a luciferase reporter construct. The promoter sequences ranged from 110 bp to 6.3 kb (1). Although these reporter constructs showed constitutive expression after transfection into several cell types, we were unable to detect any E47-dependent increase in luciferase activity (data not shown). Given that long-distance enhancers direct expression of p57Kip2 and possibly of the other genes located on the human chromosome 11p15.5 and similarly induced by E47, these results were not unexpected (see Discussion). To provide additional evidence that E47 activates transcription of the p57Kip2 gene, we asked whether two known transcriptional coactivators of E47, CBP and pCAF, can also stimulate the E47 activity towards expression of p57Kip2. Indeed, in two independent cell lines, SF210-E47-ER and U20S transiently expressing E47, the introduction of CBP and pCAF significantly potentiated E47-mediated expression of p57Kip2 (Fig. 7D and E). Taken together, these results indicate that E47 induces the expression of p57Kip2 through a transcriptional mechanism, although the cis-acting DNA elements on chromosome 11p15.5 that mediate this activation remain to be identified.

p57Kip2 is a direct target gene of E47. (A) Western blot analysis of SK-N-SH cells following transfection with pBabe-E47-ER, pBabe-bHLH-ER, or pBabe vector and treatment of the cells with 4-OHT for 24 h. (B) Northern blot analysis of SK-N-SH expressing pBabe-E47-ER and treated with CHX and 4-OHT. 28S rRNA is shown as a loading control. Western blotting for p57Kip2 shows that induction of p57Kip2 by E47-ER is fully inhibited by CHX. Asterisks indicate nonspecific bands. (C) Real-time quantitative RT-PCR for p57Kip2 and HPRT mRNAs was done from SF210-E47-ER treated with CHX and 4-OHT for the indicated times. (D) Real-time quantitative RT-PCR for p57Kip2 and HPRT mRNAs was done from SF210-E47-ER transfected with expression plasmids for CBP and pCAF and treated with 4-OHT. (E) Real-time quantitative RT-PCR for p57Kip2 and HPRT mRNAs was done from U20S transfected with the indicated combinations of expression plasmids for E47, CBP, and pCAF.

p57Kip2 is essential for cell cycle arrest induced by E47, and its expression is linked to the activity of Id2 and its negative regulator Rb in vitro and in vivo.To determine the relevance of p57Kip2 for the antiproliferative effect of bHLH transcription factors, we suppressed E47-ER-mediated up-regulation of p57Kip2 using the RNA interference technology in two different neural cell types, the neuronally derived SK-N-SH neuroblastoma cell line and the glial cell line SF210 (human glioma). In the presence of control siRNA oligonucleotides, addition of 4-OHT led to marked induction of p57Kip2 in both cell lines (Fig. 8A and B). However, transfection of cells with siRNA oligonucleotides directed against p57Kip2 abolished the E47-mediated up-regulation of p57Kip2. When the cell cycle progression of SK-N-SH and SF210 was analyzed by quantitative staining with BrdU, we found that silencing of p57Kip2 abolished the cell cycle arrest implemented by activation of E47-ER in both cell lines. This occurred in spite of a significantly different basal rate of BrdU incorporation in the two cell lines (Fig. 8C to E). To ask whether Id2 and its negative regulator Rb control the E47-mediated induction of p57Kip2, we first used the multimerized E-box-luciferase reporter construct. We confirmed that E47 robustly activated E-box transcription, and Id2 inhibited this effect. Cotransfection of unphosphorylatable, constitutively active Rb (PSM-Rb) restored E47-mediated transcriptional activation in a dose-dependent manner (Fig. 8F). In a similar experiment, we tested whether Id2 and Rb also control p57Kip2 induction by E47. We expressed E47, Id2, and PSM-Rb in the neuroblastoma cell line SK-N-SH. As expected, E47 induced the accumulation of p57Kip2 and Id2 abolished this effect. Coexpression of PSM-Rb relieved the repressive effect of Id2 and restored, although not completely, p57Kip2 up-regulation by E47 (Fig. 8G).

Induction of the expression of p57Kip2 is essential for E47-mediated cell cycle arrest of human neuroblastoma cells. SK-N-SH (A) and SF210 (B) cells expressing pBabe-E47-ER or pBabe vector were transfected with siRNA oligonucleotides expressing a scrambled sequence (CTR) or the p57Kip2 sequence (p57Kip2) before treatment with 4-OHT for 24 h. Expression of p57Kip2, α-tubulin, and Cdk4 were analyzed by Western blotting. (C) Representative fields of SK-N-SH cells treated as described for panel A. BrdU was added for 1 h prior to fixation of the cells and staining with an antibody against BrdU (red). Nuclear DNA was stained with DAPI (blue). The fractions of SK-N-SH-E47-ER (D) and SF210-E47-ER (E) incorporating BrdU after the indicated treatments were quantitated by counting at least 2,000 DAPI-positive nuclei for each duplicate transfection. (F) Quantitation of luciferase expression in SK-N-SH cells transfected with an E-box-luciferase plasmid in the presence of plasmids encoding E47, Id2, or a constitutively active, unphosphorylatable Rb (PSM-Rb). (G) SK-N-SH cells were transfected with the indicated combinations of plasmids expressing E47, Id2, and PSM-Rb. Cellular lysates were analyzed for the expression of E47, p57Kip2, Id2, PSM-Rb, and α-tubulin.

In vivo expression studies detected p57Kip2 in the embryonic mouse brain, and the expression of this CKI during development was proposed as a key factor for the decision of presumptive neurons to exit cell cycle and differentiate (8, 18, 34, 54). Given that both Id2 and E47 are expressed during neural development (23, 44, 53), an important validation of our results would be the finding that expression of Id2, a negative regulator of differentiation, is alternative to that of p57Kip2. To test this hypothesis in vitro and in vivo, we analyzed the expression of Id2 and p57Kip2 in two physiological conditions associated with cell cycle arrest and differentiation of neural cells. To recapitulate in vitro the mechanisms that drive neuroectodermal cells towards differentiation and terminal cell cycle arrest, we treated the neuroblastoma cell line LAN-1 with 1 μM retinoic acid (RA) and analyzed the expression of E47, Id2, and p57Kip2. We and others found that, when treated with pharmacologic doses of RA, LAN-1 cells undergo efficient exit from the cell cycle (25, 48). RA reduced Id2 and induced p57Kip2 in the absence of changes of E47 (Fig. 9A), suggesting that the E47:Id2 ratio is critical for E-protein-dependent activation of p57Kip2 expression. Next, we performed immunohistochemical staining for E47, p57Kip2, and Id2 in adjacent sections derived from E15.5 mouse brain. At this stage of development, neural precursors have started to migrate from the germinal area of the brain (ventricular zone [VZ]) to the postmitotic areas, which contain differentiated neurons (mantle zone [MZ]). A region with prominent expression of E47, p57Kip2, and Id2 was the inferior colliculus, a mesencephalic region located rostral to the cerebellum that, at this developmental age, displays very active neurogenesis (6, 22). Remarkably, while E47 was uniformly expressed throughout the mesencephalic wall, the pattern of expression of Id2 and p57Kip2 was clearly complementary, with Id2 being abundant in the VZ but absent in the MZ (Fig. 9B). Conversely, p57Kip2 was expressed in differentiated neurons of the MZ but was absent in the undifferentiated, proliferative area (VZ). This conclusion was further validated by double immunofluorescence for E47/p57Kip2 and Id2/p57Kip2, respectively. These experiments confirmed that E47 and p57Kip2 are coexpressed at the single-cell level in the MZ but not in the VZ, whereas Id2-positive cells in the VZ are always negative for p57Kip2 (Fig. 9C). Taken together, these results further establish the role of the E47-Id2 pathway for the regulated expression of p57Kip2 in vitro and in vivo and indicate that induction of p57Kip2 is an essential event for the antiproliferative activity of bHLH transcription factors in neural cells.

Reciprocal expression of Id2 and p57Kip2 during differentiation of neuroblastoma cells and development of the mouse brain. (A) LAN-1 cells were treated with RA, and extracts were prepared on the indicated days and analyzed by Western blotting. (B) Adjacent sections from E15.5 mouse brain were immunostained for E47, Id2, and p57Kip2. The alternative expression of Id2 and p57Kip2 in the ventricular zone (VZ) and the mantle zone (MZ) of the inferior colliculus is depicted in the right lower panel. (C) Double immunofluorescence analysis of E47 (red) and p57Kip2 (green, top panels) and Id2 (red) and p57Kip2 (green, bottom panels) from the inferior colliculus of E15.5 mouse brain. E47 and p57Kip2 colocalize in the MZ but not in the VZ, whereas cells expressing Id2 in the VZ are always p57Kip2 negative.

DISCUSSION

E2A proteins play key roles in the control of gene expression during development of many tissue types, including the nervous system. In most instances, a key aspect of the irreversible commitment to undergo a differentiation program is terminal exit from the cell cycle. Here we show that the consequence of activation of E-protein-mediated transcription in neuroectodermal cells is growth arrest, an effect caused by inhibition of entry into S phase with cell cycle block. In agreement with the pattern of expression of E proteins in the nervous system, these findings unequivocally establish the role of E proteins as antiproliferative factors that participate in terminal cell cycle arrest of neural cells primed to differentiate.

Although E2A transcription factors have been involved in the transition from proliferation to cell cycle exit (10, 13, 38) and have been reported to induce expression of several CKIs (13, 36, 39), a causal link between activation of specific target genes and cell cycle modification by E2A has never been demonstrated. Whereas E proteins may also induce other CKIs (p21Cip1, p16INK4A, p15INK4B, etc.) in specific cell types, our survey of eight human cell lines suggests that, with the exception of cells carrying hypermethylation of the p57Kip2 gene, p57Kip2 is a general target of E2A transcription factors. This observation is consistent with the notion that p57Kip2 is the only CKI required for normal development. Loss of p57Kip2 results in proliferative disorders in the lens and in cartilage as well as defects in development of several tissues (56, 58). In neuroblastoma cells, E47 caused very rapid induction of p57Kip2 mRNA and protein (2 to 4 h after activation by 4-OHT or infection by Ad-E47; Fig. 6B and 7B). Elevation of p57Kip2 preceded cell cycle arrest (evident by 8 h; Fig. 2E) and the phenotypic changes characterized by dendritic differentiation and increase of the neuronal marker MAP-2, which could be seen only after 24 h (Fig. 3B and C). Together with the finding showing that induction of p57Kip2 is essential for E47-mediated inhibition of cell cycle in neuroblastoma cells (Fig. 8), our results suggest strongly that p57Kip2 is a direct target gene recruited by bHLH transcription factors to induce quiescence of differentiated neurons. This conclusion is consistent with the general ability of p57Kip2 to arrest cell cycle progression and proliferation when ectopically expressed at moderate levels and on its own in various cell types, including those of neuroectodermal origin (27, 45, 51).

Our study established that induction of p57Kip2 by E47 does not require new protein synthesis, a feature typical of direct targets of transcription factors, and is potentiated by CBP and pCAF, two known cofactors of E47-mediated transcription (7, 9, 40). However, we have been unable to identify the region(s) in the p57Kip2 promoter/enhancer required for the activation. This is not surprising, considering that enhancers required for physiological activation of the p57Kip2 gene may lie 3′ to the gene at a distance even >250 kb from the transcriptional start site (17). In this respect, the ability of E47 to induce not only p57Kip2 but also other genes located in the chromosome 11p15.5-imprinted domain is intriguing. Indeed, the expression of each of the chromosome 11p15.5 genes that are induced by E47 (Fig. 4 and 5) is controlled by imprinting, a process marked by differential methylation of the imprinted and nonimprinted alleles. Although E47 may induce expression of p57Kip2 by reversing the status of the imprinted allele, we consider this possibility unlikely, since E47 was unable to induce p57Kip2 in cells carrying an aberrantly methylated gene (Fig. 6D). Moreover, a role for E47 in the control of methylation has never been described. We suggest that activation of distant enhancer(s), possibly acting in coordination for the p57Kip2 gene and other genes in the 11p15.5 cluster, is the most likely mechanism by which E47 induces the entire set of imprinted genes. The hypothesis that expression of multiple genes of the chromosome 11p15.5-imprinted cluster may be regulated through a common enhancer, which lies several hundred kilobases from the p57Kip2 gene, has been proposed for p57Kip2 and an adjacent gene, KvLQT1 (12, 29). Interestingly, E47 also induces the expression of KvLQT1 (data not shown). Regardless of the molecular mechanism by which E47 activates p57Kip2, the reciprocal regulation of this gene by Id2 and Rb in vitro and in vivo is an additional element supporting the idea that p57Kip2 is a direct target of bHLH transcription factors.

Although p57Kip2 has several hallmarks of a tumor suppressor gene, genetic as well as methylation-specific alterations are rarely responsible for its inactivation in neural tumors (57). Conversely, Id proteins are general effectors of oncogenic transformation in multiple tumors of neuroectodermal origin (23). We suggest that permanent inhibition of E-protein-mediated expression of p57Kip2 by deregulated Id may be a prominent mechanism driving uncontrolled proliferation and malignancy in the nervous system.

ACKNOWLEDGMENTS

We thank Paul Fisher for the immortalized human astrocytes and Rosalind John and Ben Tycko for helpful discussions.

This work was supported by grants from NIH-NCI to A.L. (R01-CA101644) and A.I. (R01-CA85628) and from the Charlotte Geyer Foundation (A.I.). G.R. was supported by a training grant from the NIH (Ruth L. Kirschstein NRSA).